Primary osteoarthritis (OA) is a musculoskeletal disorder of unknown etiology. OA is characterized by an imbalance between anabolism and catabolism in, and altered homeostasis of articular cartilage. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif are upregulated in OA joints. Extracellular matrix (ECM) proteins are critical for resistance to compressive forces and for maintaining the tensile properties of the tissue. Tissue inhibitor of metalloproteinases (TIMPs) is the endogenous inhibitor of MMPs, but in OA, TIMPs do not effectively neutralize MMP activity. Upregulation of MMP gene expression occurs in OA in a milieu of proinflammatory cytokines such as interleukin (IL)-1, IL-6 and tumor necrosis factor α. Presently, the medical therapy of OA includes mainly nonsteroidal anti-inflammatory drugs and corticosteroids which dampen pain and inflammation but appear to have little effect on restoring joint function. Experimental interventions to restore the imbalance between anabolism and catabolism include small molecule inhibitors of MMP subtypes or inhibitors of the interaction between IL-1 and its receptor. Although these agents have some positive effects on reducing MMP subtype activity they have little efficacy at the clinical level. MMP-9 is one MMP subtype implicated in the degradation of articular cartilage ECM proteins. MMP-9 was found in OA synovial fluid as a complex with neutrophil gelatinase-associated lipocalin (NGAL) which protected MMP-9 from autodegradation. Suppressing NGAL synthesis or promoting NGAL degradation may result in reducing the activity of MMP-9. We also propose initiating a search for enzyme-protein interactions to dampen other MMP subtype activity which could suppress ECM protein breakdown.

IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage.

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6 or rh-TNF-α increased the production of matrix metalloproteinase-9 (MMP-9). The effect of rhIL-6 on neutrophil gelatinase-associated lipocalin (NGAL) was also assessed. Although C28/I2 chondrocytes incubated with rhIL-6 (50 ng/ml) increased MMP-9 production which could not be mimicked by the T/C28a2 chondrocyte line, the effect of rhTNF-α on MMP-9 was more robust than with rhIL-6. The combinations of rhIL-6 and soluble IL-6 receptor-α (sIL-6Rα) or rhIL-6 and tocilizumab (TCZ), a fully-humanized recombinant monoclonal antibody that neutralizes the interaction between IL-6 and IL-6R significantly reduced MMP-9 production by C28/I2 chondrocytes. However, TCZ had no effect on rhTNF-α-induced MMP-9 production. By contrast, rhIL-6 did not increase the production of NGAL by C28/I2 chondrocytes although the number of NGAL-positive cells was significantly reduced by sIL-6R compared to its control group, but not by the combination of rhIL-6 plus TCZ compared to rhIL-6. In summary, these results showed that rhIL-6 stimulated the production of MMP-9, but not NGAL, in the C28/I2 chondrocyte line. TCZ or sIL-6Rα suppressed rhIL-6-induced MMP-9 production.

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6, a known cytokine activator of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in many cell types, caused STAT proteins to be phosphorylated. The results showed that STAT3 was constitutively phosphorylated in the absence of rhIL-6 in T/C28a2 chondrocytes. However, C28/I2 chondrocytes treated with rhIL-6 caused STAT1, STAT3, and STAT5 to be phosphorylated without altering total unphosphorylated STAT proteins. STAT3 phosphorylation in response to rhIL-6 in T/C28a and C28/I2 chondrocytes was efficiently blocked by the JAK3-selective inhibitor WHI-P131 (Janex-1) and by soluble IL-6 receptor-α (sIL-6R). However, the combination of rhIL-6 and ruxolitinib, a JAK1/JAK2-selective inhibitor, was a less effective inhibitor of STAT protein activation. These findings showed that rhIL-6 activated STAT proteins in the C28/I2 chondrocyte cell line. STAT protein phosphorylation could be blocked by a JAK3-selective inhibitor or by the combination of rhIL-6 and sIL-6R.

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We reported at the Keynote Forum of Immunology Summit-2015 that recombinant human (rh) TNF-α or rhIL-6 stimulated production of matrix metalloproteinase-9 (MMP-9) in the T/C28a2 and C-28/I2 human immortalized chondrocyte cell lines. Furthermore, we reported that tocilizumab (TCZ), a fully humanized monoclonal antibody which neutralizes IL-6-mediated signaling, inhibited the rhIL-6-mediated increase in the production of MMP-9. IL-6 is also a known activator of the JAK/STAT signaling pathway. In that regard, we evaluated the effect of rhIL-6 on total and phosphorylated Signal Transducer and Activator of Transcription by these chondrocyte lines which showed that whereas STAT3 was constitutively phosphorylated in T/C28a2 chondrocytes, rhIL-6 activated STAT3 in C-28/I2 chondrocytes. The finding that rhIL-6 increased the production of MMP-9 by human immortalized chondrocyte cell lines may have important implications with respect to the destruction of articular cartilage in rheumatoid arthritis and osteoarthritis. Thus, the markedly elevated level of IL-6 in rheumatoid arthritis and osteoarthritis sera and synovial fluid would be expected to generate significant MMP-9 to cause the degradation of articular cartilage extracellular matrix proteins. The finding that TCZ suppressed rhIL-6-mediated MMP-9 production suggests that TCZ, currently employed in the medical therapy of rheumatoid arthritis, could be considered as a drug for osteoarthritis.

T/C28a2 immortalized juvenile human chondrocytes were employed to determine the extent to which activation of Signal Transducers and Activators of Transcription-1 (STAT1) occurred in response to recombinant human interleukin-6 (rhIL-6) or rhIL-6 in combination with the soluble IL-6 receptor (sIL-6R). Two forms of STAT1, STAT1A and STAT1B, were identified on SDS-PAGE and western blotting with anti-STAT1 antibody. Western blotting revealed that STAT1 was constitutively phosphorylated (p-STAT1). Although incubation of T/C28a2 chondrocytes with rhIL-6 (50 ng/ml) increased p-STAT1A by Δ=22.3% after 30 min, this percent difference failed to reach significance by Chi-square analysis. Similarly, no effect of rhIL-6 (Δ=+10.7%) on p-STAT1B was seen at 30 min. In contrast, although the combination of rhIL-6 plus sIL-6R had no effect on p-STAT1A, rhIL-6 plus sIL-6R increased p-STAT1B by Δ=73.3% (p<0.0001) after 30 min compared to the control group and by Δ=56.7% (p<0.0001) compared to rhIL-6 alone. Janex-1, a Janus kinase-3-specific inhibitor (100 μM) partially reduced the effect of rhIL-6 on p-STAT1B by Δ=27.7% (p<0.05). The results of this study showed that STAT1A/STAT1B was constitutively activated in T/C28a2 chondrocytes. Although rhIL-6 increased p-STAT1B to a small extent, the combination of rhIL-6 plus sIL-6R was far more effective in stimulating STAT1B phosphorylation compared to controls or rhIL-6 alone. These data support the likelihood that although JAK3-mediated activation of STAT1 in T/C28a2 chondrocytes may involve the IL-6/IL-6R/gp130 pathway, these results indicated that STAT1 activation in response to IL-6 preferentially involved IL-6 trans-signaling via sIL-6R.

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